Cloning of mite allergens

ABSTRACT

Isolated DNA encoding allergens of Dermatophagoides (house dust mites) particularly of the species Dermatophagoides farinae and Dermatophagiodes pteronyssinus, which are protein allergens or peptides which include at least one epitope of the protein allergen. In particular, DNA encoding two major D. farinae allergens, Der f I and Der f II and DNA encoding a D. pteronyssinus allergen, Der p II. In addition, the proteins or peptides encoded by the isolated DNA, their use as diagnostic and therapeutic reagents and methods of diagnosing and treating sensitivity to house dust mite allergens.

This application is a continuation application of Ser. No. 08/162,722 filed on Nov. 8, 1993, pending, which in turn is a continuation application of Ser. No. 07/458,642 filed on Feb. 13, 1990, abandoned. This application claims priority to PCT/AU88/00195 filed on Jun. 17, 1988 which claims priority to Australian Patent Application No. PI 2523/87 filed on Jun. 18, 1987. The contents of all of the aforementioned application(s) are hereby incorporated by reference.

This invention relates to the cloning of site allergens, in particular it relates to the cloning and expression of DNA coding for house dust mite allergens.

It has long been recognised that the allergy to mites of the genus Dermatophagoides is associated with diseases such as asthma, rhinitis, and atopic dermatitis (13,14). In this regard, the species D. pteronyssinus and D. farinae predominate and many studies have been performed to identify the allergens they produce. Progress in studying allergy to the house dust mite has been hindered by difficulties in obtaining standardised allergens (1). About 6 antigens elicit most IgE anti-mite antibody responses (2,3) and one of these, Der p I, an approx. 27 kDa glycoprotein found in mite faeces reacts with about 80-90% of allergic sera usually as the predominant antigen. Also, because it is a constituent of faeces it is readily airborne and solubilised (4), thus it is presented in a form which is more accessible than allergens in mite bodies.

From 5-10% of people in most populations suffer from asthma, a condition often closely associated with allergy to the house dust mite, Dermatophagoides pteronyssinus. This type of allergy is mediated by IgE antibodies which react to mite components. The possibility, therefore, exists that desensitisation therapy would be successful for asthma in much the same way as a similar allergy to bee sting can be desensitised by repeated and progressively increased doses of bee venom.

It is therefore one object of the present invention to provide a method for the production of house dust mite allergens, particularly the Der p I and Der p II allergen, suitable for use in such desensitisation therapy as well as in diagnostic tests for the detection of allergy.

According to the present invention, there is provided a recombinant DNA molecule comprising a nucleotide sequence capable of being expressed as a polypeptide displaying the antigenicity of an allergen of house dust mites of the genus Dermatophagoides, or a peptide comprising at least one epitope thereof, or a nucleotide sequence which hybridizes with such a sequence under conditions of high stringency. In one particular embodiment the nucleotide sequence is capable of being expressed as the major Der p I or the Der p II allergen of the house dust mite, or a peptide comprising at least one epitope thereof. By way of example, the nucleotide sequence may be characterised as having substantially all or part of the sequence shown in FIG. 1 or FIG. 7.

In work leading to the present invention, recombinant DNA molecules have been constructed comprising nucleotide sequences which code for expression or the Der p I and Der p II allergens from Dermatophagoides pteronyssinus. The species Dermatophagoides farinae and Dermatophagoides microcerus, as well as another house dust mite of the same family, Euroglyphus maynei, produce similar allergens with antigenic cross reactivity and a high degree of amino acid homology. Thus the present invention extends not only to allergens from Dermatophagoides pteronyssinus but also to other mite allergens coded for by DNA which hybridises with its Dermatophagoides pteronyssinus counterpart under conditions of high stringency.

In another aspect, this invention provides an expression vector, such as a bacteriophage, containing a nucleotide sequence as described above, and an expression control sequence operatively linked thereto. In addition, this invention provides a host cell containing an expression vector as described above.

In yet another aspect, this invention provides a synthetic protein or polypeptide displaying the antigenicity of all or a portion of an allergen of house dust mites of the genus Dermatophagoides, in particular the major Der p I or Der p II allergens, or allergens from other house dust mites with antigenic cross-reactivity and a high degree of amino acid homology. Such a synthetic product may comprise a fusion protein comprising a polypeptide component displaying the antigenicity of all or a portion of an allergen of the house dust mite and fused thereto an additional polypeptide , such as β-galactosidase, coded for by the DNA of an expression vector.

A method for the production of a synthetic protein or polypeptide as described above, in particular a fusion protein as described, comprises the steps of culturing a host cell containing an expression vector as described, and recovering the synthetic protein or polypeptide from the culture.

The term "synthetic" as used herein relates to proteins or polypeptides produced by cloning and expression of a nucleotide sequence as described herein.

The synthetic proteins or polypeptides or "synthetic allergens" produced in accordance with this invention display the antigenicity of the native allergen. By way of example, a cDNA clone coding for the major allergen Der p I produces a fusion protein which reacts with rabbit anti-Der p I antiserum. Accordingly, such a fusion protein has potential for use as an antigenic component in diagnostic tests and therapeutic (for example desensitisation) compositions and treatments.

In particular, the synthetic allergens may be used as in vitro and in vivo diagnostic reagents as demonstrated by their antigenic similarity with the native mite allergens and their ability to react with IgE from serum of people with allergy to mites. Further, they may be used as therapeutic substances by desensitisation as demonstrated by the ability of synthetic Der p I to inhibit IgE responses of mice to mature Der p I.

Further features of the present invention will be apparent from the following Example which is included by way of illustration of the invention. In the drawings:

FIGS. 1A & 1B show the nucleotide and predicted amino acid sequence of cDNA λgt 11 p1(13T). Numbers to the right are nucleotide positions whereas numbers above the sequence are amino acid positions. Positive amino acid residue numbers correspond to the sequence of the mature excreted Der p I beginning with threonine. Negative sequence numbers refer to the proposed transient pre- and preproenzyme forms of Der p I. The arrows indicate the beginning of the proposed proenzyme sequence and the mature Der p I, respectively. Residues -15 to -13 enclosed by an open box make up the proposed cleavage for the proenzyme formation, and the dashed residues 52-54 represent a potential N-glycosylation site. The termination TAA codon and the adjacent polyadenylation signal are underlined. Amino acid residues 1-41, 79-95, 111-142, and 162-179 correspond to known tryptic peptide sequences determined by conventional amino acid sequencing analysis.

FIG. 2 shows the restriction map of the cDNA insert of clone λgt11 p1(13T) and the strategy of DNA sequencing. Arrows indicate directions in which sequences were read.

FIG. 3 is a comparison of N-terminal sequences of Der p I and Der f I. The amino acid sequence for Der p I is equivalent to amino acids 1-20 in FIG. 1; the Der f I sequence is from reference (12).

FIG. 4 shows the reactivity of λgt11 p1(13T) with anti Der p 1. Lysates from Y1089 lysogens induced for phage were reacted by dot-blot with rabbit anti-Der p 1 (Der p I) or normal rabbit serum (Nrs). Dots (2 μl) were made in triplicate from lysates of bacteria infected with λgt11 p1(13T) (a) or λgt11 (b). When developed with ¹²⁵ I-protein A and autoradiography only the reaction between λgt11 p1(13T) lysate and the anti-Der p 1 showed reactivity.

FIG. 5 shows reaction of clone pGEX-pl(13T) with IgE in allergic serum. Overnight cultures of PGEX or pGEX-p1 where diluted 1/10 in broth and grown for 2 hours at 37°. They were induced with IPTG, grown for 2 hours at 37°. The bacteria were pelletted and resuspended in PBS to 1/10 the volume of culture media. The bacteria were lysed by freeze/thaw and sonication. A radioimmune dot-blot was performed with 2 μl of these lysates using mite-allergic or non-allergic serum. The dots in row 1 were from E. coli containing pGEX and row 2-4 from different cultures of E. coli infected with pGEX-p1(13T). Reactivity to pGEX-p1 (13T) was found with IgE in allergic but not non-allergic serum. No reactivity to the vector control or with non-allergic serum was found.

FIG. 6 shows seroreactivity of cDNA clones coding for Der p II in plaque radioimmune assay. Segments of nitrocellulose filters from plaque lifts were taken from clones 1, 3, A, B and the vector control Amp1. These were reached by immunoassay for human IgE against allergic serum (AM) in row 1, non-allergic serum (WT) in row 2 and by protein A immunoassay for Der p I with rabbit antiserum in row 3. The clones 1, 3 and B reacted strongly with allergic serum but not non-allergic or vector control. (Clone B and vector control were not tested with non-allergic serum).

FIGS. 7A & 7B show the nucleotide and predicted amino acid sequence of cDNA of λgt11 p II (Cl). Numbers to the right are nucleotide positions and numbers above are amino acid positions. Positive numbers for amino acids begin at the known N-terminal of Der p II and match the known sequence of the first 40 residues. Residues -1 to -16 resemble a typical leader sequence with a hydrophobic core.

FIG. 8 shows the N-terminal amino acid homology of Der p II and Der f II. (Der f II sequence from reference 30.)

EXAMPLES 1 Materials and Methods

Cloning and Expression of cDNA.

Polyadenylated mRNA was isolated from the mite Dermatophagoides pteronyssinus cultured by Commonwealth Serum Laboratories, Parkville, Australia, and cDNA was synthesised by the RNA-ase H method (5) using a kit (Amersham, International, Bucks). After the addition of EcoRI linkers the cDNA was ligated into λgt11 and plated in E. coli Y1090 (r-) (Promega Biotec, Madison, Wis.), to produce a library of 5×10⁵ recombinants. Screening was performed by plaque radioimmune assay (6) using a rabbit anti-Der p 1 antiserum (7). Reactivity was detected by developing with ¹²⁵ I-Staphyloccocal protein A and autoradiography (8). Phage producing plaques reactive with the antiserum were checked for the ability of their DNA to hybridise with 17-mer oligonucleotides constructed on the basis of amino acid sequences obtained from three tryptic peptides obtained from faecal Der p 1 (17).

In brief, Der p 1 was isolated by gel filtration, chromatofocusing and fast liquid protein chromatography on a TSK 3000 SW column and then high performance reversed-phase liquid chromatography. Reduction, carboxymethlation and tryptic digestion were performed by standard techniques (9) and peptides were isolated by HPLC using a Brownlee Bu-300 column and a linear gradient of 0-60% A-B where A was 10 mM KH₂ PO₄ pH6.5, and B was acetonitrile. Amino acid sequences were determined on an Applied Biosystems (Model 470A) gas phase sequencer (9). Oligonucleotides were synthesised using an Applied Biosystem model 370A synthesiser and purified by reversed-phase HPLC. For hybridigation, oligonucleotides were labelled by polynucleotide kinase (Promega Biotec) using 20 pmole of DNA and 20 pmole γ-32P-ATP at 1400 Ci/mmole (10). Labelled oligonucleotides were purified by 15% polyacrylamide gel electrophoresis. Plaques were lifted onto nitrocellulose (Schleicher and Schull, Dassel, West Germany) and denatured and baked (10). Hybridisations were performed overnight in 6× sodium chloride sodium citrate pH 7 (SSC) (10), 0.1% SDS at 37° at 10⁶ cpm/ml and washed for 1 hr. at 37° C. in 6× SSC containing 0.1% Triton X-100.

Isolation of Genomic DNA.

Extraction of mite genomic DNA was carried out by a modified guanidium-HCl/cesium chloride (CsCl) method (15). 10 g of live mites were ground in the presence of liquid nitrogen and sand to form a paste. 8 ml of 6 M guanidine hydrochloride in 0.1 M sodium acetate buffer pH 5.2 were then added and the mixture was homogenized and spun at 10,000 rpm for 30 min in a Sorval SS34 rotor. The supernatant was collected and layered onto a CsCl pad (5 ml of 4.8 M CsCl in 10 mM EDTA) and centrifuged at 37,000 rpm for 16 h at 15° C. in a SW41 T1 rotor (Beckman Instruments, Inc., Fullerton, Calif.). The DNA band at the interphise was collected and diluted 1:15 in 10 mM Tris HCl/1 mM EDTA buffer, pH 8.0. Banding of genomic DNA in CsCl was carried out by the standard method.

Isolation of DNA from λgt11 p1 cDNA Clone.

Phage DNA from λgt11 p1 clone was prepared by a rapid isolation procedure. Clarified phage plate lysate (1 ml) was mixed with 270 μl of 25% wt/vol polyethylene glycol (PEG 6000) in 2.5 M NaCl and incubated at room temperature for 15 min. The mixture was then spun for 5 min in a microfuge (Eppendorf, Federal Republic of Germany), and the supernatant was removed. The pellet was dissolved in 100 μl of 10 mM Tris/HCl pH 8.0 containing 1 mN EDTA and 100 mM NaCl. This DNA preparation was extracted 3 times with phenol/chloroform (1:1) and the DNA was precipitated by ethanol.

DNA Hybridization.

Nucleic acid was radiolabelled with ³² P by nick translation (10). DNA samples were digested with appropriate restriction enzymes using conditions recommended by the supplier. Southern blots were prepared using Zeta-Probe membranes (Bio-Rad Laboratories, Richmond, Calif.). Prehybridization, hybridization, posthybridization washes were carried out according to the manufacturers recommendations (bulletin 1234, Bio-Rad Laboratories).

Cloning and DNA Sequencing.

To clone the 0.8-kb cDNA insert from clone λgt11 p 1 into plasmid pUC8, phage DNA was digested with EcoRI restriction enzyme and then ligated to EcoRI-digested pUC8 DNA and used to transform Escherichia coli JM83. The resulting recombinant plasmid was designated as pHDM 1.

To obtain clones for DNA sequence analysis, the cDNA insert was isolated from pHDM 1 and ligated to M13-derived sequencing vectors mp18 and mp19 (16). Transformation was carried out using E. coli JM107 and sequencing was performed by the dideoxynucleotide chain termination method (11).

RESULTS

Several phage clones reacted with the rabbit anti Der p I serum and hybridised with all 3 oligonucleotide probes. One of these, λgt11 p1(13T), was examined further. The nucleotide sequence of the cDNA insert from this clone, λgt11 p 1, was determined using the sequencing strategy shown in FIG. 2. The complete sequence was shown to be 857 bases long and included a 69-base-long 5' proximal end sequence, a coding region for the entire native Der p I protein of 222 amino acids with a derived molecular weight of 25,371, an 89-base-long 3' noncoding region and a poly (A) tail of 33 residues (FIG. 1).

The assignment of threonine residue at position 1 as the NH₂ -terminal amino acid of Der p I was based on data obtained by NH₂ -terminal amino acid sequencing of the pure protein isolated from mite excretions (17). The predicted amino acid sequence matched with data obtained by amino acid sequence analysis of the NH₂ -terminal region as well as with internal sequences derived from analyses of tryptic peptides (FIG. 1). The complete mature protein is coded by a single open reading frame terminating at the TAA stop codon at nucleotide position 736-738. At present, it is not certain whether the first ATG codon at nucleotide position 16-18 is the translation initiation site, since the immediate flanking sequence of this ATG codon (TTGATGA) showed no homology with the Kozak consenses sequence (ACCATGG) for the eukaryotic translation initiation sites (18). In addition, the 5' proximal end sequence does not code for a typical signal peptide sequence (see below).

The amino acid sequence predicted by nucleotide analysis is shown in FIG. 1. A protein data-base search revealed that the Der p I amino acid sequence showed homology with a group of cysteine proteases. Previous cDNA studies have shown that lysosomal cathepsins B, a mouse macrophage protease and a cysteine protease from an amoeba have transient pre- and proform intermediates (19-21), and inspection of the amino acid sequence at the 5' proximal end of the λgt11 p1 cDNA clone suggests that Der p I may be similar. First, the hydrophilicity plot (22) of the sequence preceding the mature protein sequence lacks the characteristic hydrophobic region of a signal peptide (23) and second, an Ala--X--Ala sequence, the most frequent sequence preceding the signal peptidase cleavage site (24,25), is present at positions -13, -14, -15 (FIG. 1). Therefore, it is proposed that cleavage between pro-Der p I sequence and the pre-Der p I sequence occurs between Ala (-13) and Phe (-12). Thus, pro-Der p I sequence begins at residues Phe (-12) and ends at residues Glu (-1). The amino acids residues numbered -13 to -23 would then correspond to a partial signal peptide sequence.

When the 857-bp cDNA insert was radiolabelled and hybridized against a Southern blot of EcoRI-digested genomic DNA from house dust mite, hybridization to bands of 1.5, 0.5, and 0.35 kb was observed (data not shown). As shown in the restriction enzyme map of the cDNA insert (FIG. 2), there was no internal EcoRI site and the multiple hybridization bands observed suggest that Der p I is coded by a noncontiguous gene. The results also showed little evidence of gene duplication since hybridization was restricted to fragments with a total length of 2.4 kb.

The N-terminal can be compared with N-terminal of the equivalent protein from D. farinae (Der f 1) (12). There is identity in 11/20 positions of the sequences available for comparison (FIG. 3).

To examine the protein produced by λgt11 p1(13T), phage was lysogenised in Y1089 (r-) and the bacteria grown in broth culture at 30°. Phage was induced by temperature switch and isopropyl thiogalactopyranoside (IPTG) (6) and the bacteria were suspended in PBS to 1/20 of the culture volume, and sonicated for an antigen preparation. When examined by 7.5% SDS-PAGE electrophoresis it was found that λgt11 p1(13T) did not produce a Mr 116K β-galactosidase band but instead produced a 140K band consistent with a fusion protein with the Der p I contributing a 24 kDa moiety (6). Rabbit anti Der p I was shown to react with the lysate from λgt11 p1(13T) (FIG. 4).

EXAMPLE 2

Expression of Der p I cDNA Products Reactive With IgE From Allergic Serum.

The DNA insert from λgt11 p1(13T) which codes for Der p I was subcloned into the EcoRI site of the plasmid expression vector (pGEX)(26) where it could be expressed as a fusion with a glutathione transferase molecule. E. coli infected with this plasmid pGEX-p1(13T) or with the vector alone were grown to a log phase culture and harvested by centrifugation. The bacteria were suspended in PBS to 1/20 of their culture volume and lysed by freeze- thawing. The lysate was shown by sodium didodecyl- sulphate polyacrylamide electrophoresis to express a fusion protein in high concentration of the expected Mr 50,000. These lysates were then tested for their ability to react with IgE from allergic serum by radioimmune dot-blot conducted by the method described by Thomas and Rossi (27). The serum was taken from donors known to be mite-allergic or from non-allergic controls. Reactivity was developed by ¹²⁵ I-monoclonal anti-IgE and autoradiography. FIG. 5 shows the lysate from pGEX-p1(13T), but not the vector control reacted with IgE in allergic serum, but not non allergic serum.

EXAMPLE 3

Inhibition of IgE Antibody Responses to Der p I by Treatment With the Product From a cDNA Clone Coding for Der p I.

E. coli lysogenised by λgt11 p1(13T) were grown and induced by tempereature switch to produce a recombinant fusion protein which was consistent with a 24 kd Der p I moiety and a 116 kd β⁴ -galactosidase moiety (p1(13T) (28). This protein was mostly insoluble and could be isolated to about 90% purity, judged by sodium didodecyl polyacrylamide electrophoresis, by differential centrifugation. A similar protein was produced from another gt11 cDNA mite clone λgt pX (2c). To test for the ability of the recombinant protein to modify IgE antibody responses to Der p I, groups of 4-5 CBA mice were injected intraperitoneally with 2 mg of the p1 (13T) or pX (2c) fusion proteins and after 2 days given a subcutaneous injection of 5 μg of native Der p I (from mite culture medium) in aluminium hydroxide gel. The IgE antibody titres were measured by passive cutaneous anaphylazis (PCA) after 3 and 6 weeks. The methods and background data for these responses have been described by Stewart and Holt (29). For a specificity control, groups of mice injected with pl(13T) or pX (2c) were also injected with 10 μg of ovalbumin in alum. Responses were compared to mice without prior p1(13T) or pX (2c) treatment (Table 1). After 3 weeks mice either not given an injection of recombinant protein or injected with the control pX (2c) had detectable anti Der p I PCA titres (1/2 or greater). Only 1/5 of mice treated with recombinant p1(13T) had a detectable titre and this at 1/4 was lower than all of the titres of both control groups. Titres of all groups at 6 weeks were low or absent (not shown). The PCA response to ovalbumin was not significantly affected by treatment with recombinant proteins. These data show the potential of the recombinant proteins to specifically decrease IgE responses as required for a desensitising agent.

                  TABLE 1                                                          ______________________________________                                         Inhibition of anti Der p I IgE by preinjection with                             recombinant Der p I.                                                                           immunising                                                                                               preinjection injection (d0)                                                   IgE (PCA) titres at d21              group -2 days    (5 μg/alum)                                                                            responders                                                                              titres                                    ______________________________________                                         1     --         Der p I    4/4      1/16-1/64                                   2 pX(2C) Der p I  5/5 1/8-1/16                                                 3 p1(13T) Der p I   1/5* 1/4*                                                  4 -- ovalbumin 4/4 1/64-1/256                                                  5 pX(2C) ovalbumin 5/5 1/32-1/128                                              6 p1(13T) ovalbumin 5/5 1/64-1/256                                           ______________________________________                                          Mice were given a preinjection on day -2 and then immunised with Der p I       or ovalbumin on day 0. Serum antibody titres were measured on day 21 and       42 by PCA in rat skin. Significant anti Der p I titres were not detected       on day 42 (not shown). The PCA were measured to Der p I for groups 1-3 an      ovalbumin for groups 4-6. The anti Der p I titres were lower (p < 0.001)*      when pretreated with recombinant Der p I p1(13T).                              *Mann Whitney analysis.                                                  

EXAMPLE 4

Expression of Der p I Antigenic Determinants by Fragments of the cDNA From λgt11 p1(13T)

The cDNA from λgt11 (13T) coding for Der p I was fragmented by sonication. The fragments (in varying size ranges) were isolated by electrophoresis, filled in by the Klenow reaction to create blunt ends. EcoRI linkers were attached and the fragment libraries cloned in λgt11. The methods used for the fragments cloning were the same as that used for cDNA cloning (6). Plaque immunoassay was used for screening with rabbit anti Der p I. Three phage clones reacting with the antiserum were isolated and the oligonucleotide sequences of the cloned fragments obtained. Two of these were found to code for Der p I amino acids 17-55 (see FIG. 1 for numbering) and one for amino acids 70-100. Such fragments will eventually be useful for both diagnostic reagents to determine epitope reactivity and for therapy where molecules of limited allergenicity may increase safety of desensitisation.

EXAMPLES 5

Cloning and Expression of cDNA Coding for the Major Mite Allergen Der p II.

The Dermatophagoides pteronyssinus cDNA library in λgt11 previously described was screened by plaque radioimmune assay using nitrocellulose lifts (6). Instead of using specific antisera the sera used was from a person allergic to house dust mites. The serum (at 1/2 dilution) was absorbed with E. coli. To detect reactivity an ¹²⁵ I labelled monoclonal anti IgE was used (at 30 ng/ml with 2×10⁶ cpm/ml (approx. 30% counting efficiency)). After 1 hour the filters were washed and autoradiography performed. Using this procedure 4 clones reacting with human IgE were isolated. It was found they were related by DNA hybridisation and had an identical pattern of reactivity against a panel of allergic sera. FIG. 6 shows IgE reactivity in plaque radioimmunoassay against allergic serum (AM) (top row) or non allergic (WT). Here clones 1, 3 and 8 react strongly, but only against allergic sera. The amp 1 segments (present in row 1) are a λgt11 vector control. The bottom row is an immunoassay with rabbit anti Der p I, developed by ¹²⁵ I staphylococcus protein A which shows no significant reactivity. The clones were tested against a panel of sera. Serum from five patients without allergy to mite did not react, but serum from 14/17 people with mite allergy showed reactivity. The DNA insert from the clone λgt11 pII(C1) was subcloned into M13 mp18 and M13 mp19 and sequenced by the chain termination method. The nucleotide sequence (FIG. 7) showed this allergen was Der p II by (a) The homology of the inferred amino acid sequence of residue 1-40 with that of the N-terminal amino acid of Der p II (30) and (b) the homology of this sequence with the equivalent Der f II allergen from Dermatophagoides farinae (30).

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We claim:
 1. An isolated nucleic acid comprising a nucleotide sequence which hybridizes under high stringency conditions to the nucleotide sequence shown in FIG. 7, wherein said high stringency conditions comprise hybridization at Tm-20 followed by at least one post-hybridization wash at Tm-12.
 2. An isolated nucleic acid encoding a Der p II protein allergen, said nucleic acid comprising the nucleotide sequence shown in FIG. 7 or the coding region thereof.
 3. An isolated nucleic acid comprising a nucleotide sequence which encodes a Der p II protein allergen having the amino acid sequence shown in FIG. 7 or the mature portion thereof. 